Our recent identification and preliminary characterization of a lymphatic vascular system in the zebrafish has made it possible to bring the powerful genetic, experimental embryologic,and imaging tools available in this model organism to bear on the question of how lymphatic vessels form and what regulates their growth and assembly. Understanding how to regulate and control lymphatic vessel formation is a topic of considerable clinical interest given recent evidence suggesting that lymphatics are the major route for tumor metastasis in many if not most cancers. The long-term goal of this project is to expand on our preliminary findings in order to gain a more detailed understanding of how lymphatic vessels assemble in the fish, and then use this powerful model to study how lymphangiogenesis is regulated in vivo. The lymphatic system has become the subject of great interest in recent years because of its important role in normal and pathological processes, but progress in understanding the origins and early development of this system has been hampered by difficulties in observing lymphatic cells in vivo and performing defined genetic and experimental manipulation of the lymphatic system in currently available model organisms. We recently showed for the first time that the zebrafish possesses a lymphatic system that shares many of the morphological, molecular, and functional characteristics of the lymphatic vessels found in other vertebrates (including humans), providing a superb new model for imaging and studying lymphatic development. Using two-photon time-lapse imaging of transgenic zebrafish, we also traced the migration and lineage of individual cells incorporating into the lymphatic endothelium, providing the first conclusive in vivo evidence establishing that early lymphatic endothelial cells are derived from primitive venous blood vessels. We are continuing to examine the assembly and origins of the lymphatic system of the zebrafish. In ongoing studies we are using time-lapse two-photon imaging of transgenic animals, lymphangiography (imaging of dye injected into lymphatic vessels), histology, and scanning and transmission electron microscopy to further characterize how the lymphatic vascular system grows and assembles during development. We are also examining the spatial and temporal expression of lymphatic genes to gain a better understanding of where and how cells become specified as lymphatic endothelial cells. In addition, we are characterizing a number of novel as well as previously characterized genes. Our goal is to determine what functional role they play in lymphatic endothelial specification and differentiation, and in lymphatic vessel formation and growth. We have identified several lymphatic specific mutants in genetic screens and are now carrying out positional cloning and characterization of these mutants. We are also using antisense oligonucleotide knockdown technologies to examine the loss-of-function phenotypes of a number of genes. As one example of the results of these studies we have recently used these knockdown methods to show that netrin, a secreted molecule previously shown to have an important role in neuronal guidance, is also required for lymphatic assembly and patterning. The phenotypes of mutants and antisense oligonucleotide-treated embryos are being assessed by imaging lymphatic vessels in transgenic animals and examining expression of lymphatic marker genes. The results of our studies, combining the genetic and experimental tools available in the zebrafish with the ability to perform high-resolution microscopic imaging of developing vascular structures in living animals, are leading to important new insights into the origins and assembly of the lymphatic system.